Analytical and Bioanalytical Chemistry

, Volume 384, Issue 1, pp 286–294 | Cite as

Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit components and application to grain samples

  • Anna Yu Kolosova
  • Won-Bo Shim
  • Zheng-You Yang
  • Sergei A. Eremin
  • Duck-Hwa Chung
Original Paper


A direct competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody has been developed and optimized for detection of aflatoxin B1 (AFB1), and an ELISA kit has been designed. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for aflatoxin monitoring. AFB1 concentrations determinable by ELISA ranged from 0.1 to 10 μg L−1. The IC50 value was 0.62 μg L−1. Recovery from spiked rice samples averaged between 94 and 113%. The effect of different reagents on the stability of HRP–AFB1 conjugate solution was studied. The performance of a stabilized enzyme tracer in ELISA was determined and compared with that of a freshly prepared control solution of HRP–AFB1 conjugate. The results showed that stabilizing media containing 0.02% BSA, 0.1% Kathon CG, and 0.05 mol L−1 calcium chloride in 0.05 mol L−1 Tris-HCl buffer (pH 7.2) maintained the activity of HRP–AFB1 at a dilution of 1:1000 for a period of at least 12 months at room temperature whereas the reference conjugate solution without the additives lost its activity within a few days. Several additives were tested for their stabilizing effect on a monoclonal antibody (MAb) immobilized on the surface of polystyrene microtitre plates. It was shown that immobilized MAb, treated with post-coating solutions containing PVA, BSA, and combinations of these substances with trehalose, retained its activity for at least 4 months at 4°C, whereas the untreated MAb-coated plate lost its activity within 2 days.


ELISA Monoclonal antibodies Aflatoxin B1 HRP conjugate Stabilization 



This research was partially supported by the National Toxicology Program of the National Institute of Toxicological Research, Brain Korea 21 Program from the Ministry of Education and the Environmental Biotechnology National Core Research Centre (grant R15-2003-012-01001-0) from KOSEF/MOST, Korea.


  1. 1.
    International Agency for Research on Cancer (1993) IARC monograph on the evaluation of carcinogenic risk to humans, vol 56. IARC, Lyon, FranceGoogle Scholar
  2. 2.
    Food and Agriculture Organisation of the United Nations (1995) FAO Food and Nutrition Paper. FAO, Rome, ItalyGoogle Scholar
  3. 3.
    European Commission (1998), Commission Regulation 1525/98 Off J L201, 0093Google Scholar
  4. 4.
    Scott PM (1993) Trends Anal Chem 12(9):382–386CrossRefGoogle Scholar
  5. 5.
    Stroka J, van Otterdijk R, Anklam E (2000) J Chromatogr A 904:251–256CrossRefPubMedGoogle Scholar
  6. 6.
    Jaimez J, Fente CA, Vazquez BI, Franco CM, Cepeda A, Mahuzier G, Prognon P (2000) J Chromatogr A 882:1–10CrossRefPubMedGoogle Scholar
  7. 7.
    Papp E, Otta KH, Záray G, Mincsovics E (2002) Microchem J 73:39–46CrossRefGoogle Scholar
  8. 8.
    Otta KH, Papp E, Bagócsi B (2000) J Chromatogr A 882:11–16CrossRefPubMedGoogle Scholar
  9. 9.
    Escobar A, Regueiro OS (2002) J Food Prot 65(1):219–221PubMedGoogle Scholar
  10. 10.
    Park JW, Kim EK, Kim YB (2004) Food Addit Contam 21(1):70–75CrossRefPubMedGoogle Scholar
  11. 11.
    Lee NA, Wang S, Allan RD, Kennedy IR (2004) J Agric Food Chem 52:2746–2755CrossRefPubMedGoogle Scholar
  12. 12.
    Roch OG, Blunden G, Coker RD, Nawaz S (1995) Food Chem 52:93–98CrossRefGoogle Scholar
  13. 13.
    Holcomb M, Thompson HC Jr, Cooper WM, Hopper ML (1996) J Supercrit Fluids 9:118–121CrossRefGoogle Scholar
  14. 14.
    Elizalde-González MP, Mattusch J, Wennrich R (1998) J Chromatogr A 828:439–444CrossRefPubMedGoogle Scholar
  15. 15.
    Sharma M, Márquez C (2001) Animal Feed Sci Technol 93:109–114CrossRefGoogle Scholar
  16. 16.
    Blesa J, Soriano JM, Moltó JC, Marín R, Maòes J (2003) J Chromatogr A 1011:49–54CrossRefPubMedGoogle Scholar
  17. 17.
    Chiavaro E, Dall'Asta C, Galaverna G, Biancardi A, Gambarelli E, Dossena A, Marchelli R (2001) J Chromatogr A 937:31–40CrossRefPubMedGoogle Scholar
  18. 18.
    Peòa R, Alcaraz MC, Arce L, Ríos A, Valcárcel M (2002) J Chromatogr A 967:303–314CrossRefPubMedGoogle Scholar
  19. 19.
    Marrazza G, Chianella I, Mascini M (1999) Anal Chim Acta 387:297–307CrossRefGoogle Scholar
  20. 20.
    Maragos CM, Thompson VS (1999) Nat Toxins 7:371–376CrossRefPubMedGoogle Scholar
  21. 21.
    Van der Gaag B, Spath S, Dietrich H, Stigter E, Boonzaaijer G, van Osenbruggen T, Koopal K (2003) Food Control 14:251–254CrossRefGoogle Scholar
  22. 22.
    Devi KT, Mayo MA, Reddy KLN, Delfosse P, Reddy G, Reddy SV, Reddy DVR (1999) Lett Appl Microbiol 29:284–288CrossRefPubMedGoogle Scholar
  23. 23.
    Gathumbi JK, Usleber E, Märtlbauer E (2001) Lett Appl Microbiol 32:349–351CrossRefPubMedGoogle Scholar
  24. 24.
    Korde A, Pandey U, Banerjee S, Sarma HD, Hajare S, Venkatesh M, Sharma AK, Pillai MRA (2003) J Agric Food Chem 51:843–846CrossRefPubMedGoogle Scholar
  25. 25.
    Garden SR, Strachan NJC (2001) Anal Chim Acta 444:187–191CrossRefGoogle Scholar
  26. 26.
    Daly SJ, Keating GJ, Dillon PP, Manning BM, O'Kennedy R, Lee HA, Morgam MRA (2000) J Agric Food Chem 48:5097–5104CrossRefPubMedGoogle Scholar
  27. 27.
    Ho J-AA, Wauchope RD (2002) Anal Chem 74:1493–1496CrossRefPubMedGoogle Scholar
  28. 28.
    Pal A, Dhar TK (2004) Anal Chem 76:98–104CrossRefPubMedGoogle Scholar
  29. 29.
    Nasir MS, Jolley ME (2002) J Agric Food Chem 50:3116–3121CrossRefPubMedGoogle Scholar
  30. 30.
    Bhattacharya D, Bhattacharya R, Dhar TK (1999) J Immunol Methods 230:71–86CrossRefPubMedGoogle Scholar
  31. 31.
    Lipigorngoson S, Limtrakul P, Suttajit M, Yoshizawa T (2003) Food Addit Contam 20(9):838–845CrossRefPubMedGoogle Scholar
  32. 32.
    Gathumbi JK, Usleber E, Ngatia TA, Kangethe EK, Märtlbauer E (2003) Poult Sci 82(4):585–590PubMedGoogle Scholar
  33. 33.
    Ó'Fágáin C (1995) Biochim Biophys Acta 1252:1–14PubMedGoogle Scholar
  34. 34.
    Ó'Fágáin C (2003) Enzyme Microb Technol 33:137–149CrossRefGoogle Scholar
  35. 35.
    Schütz AJ, Winklmair M, Weller MG, Niessner R (1997) SPIE 3105:332–340CrossRefGoogle Scholar
  36. 36.
    Kühlmeyer C, Klein J (2002) Enzyme Microb Technol 6196:1–8Google Scholar
  37. 37.
    Dankwardt A, Müller J, Hock B (1998) Anal Chim Acta 362:35–45CrossRefGoogle Scholar
  38. 38.
    Eremin AN, Budnikova LP, Sviridov OV, Metelitsa DI (2002) Appl Biochem Microbiol 38(2):151–158CrossRefGoogle Scholar
  39. 39.
    Chu FS, Hsia MTS, Sun PS (1977) J AOAC Int 60:791–794Google Scholar
  40. 40.
    Langone JJ, Van Vunakis H (1975) Res Commun Chem Pathol Pharmacol 10:163–171PubMedGoogle Scholar
  41. 41.
    Kolosova AYu, Park JH, Eremin SA, Kang SJ, Chung DH (2003) J Agric Food Chem 51:1107–1114CrossRefPubMedGoogle Scholar
  42. 42.
    Heiss C, Weller MG, Niessner R (1999) Anal Chim Acta 396:309–316CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Anna Yu Kolosova
    • 1
    • 2
    • 3
  • Won-Bo Shim
    • 1
  • Zheng-You Yang
    • 1
  • Sergei A. Eremin
    • 2
  • Duck-Hwa Chung
    • 1
  1. 1.Laboratory of Food Analysis, Group of Bioanalysis, Faculty of Pharmaceutical SciencesGhent UniversityGhentBelgium
  2. 2.Division of Chemical Enzymology, Faculty of ChemistryM.V. Lomonosov Moscow State UniversityMoscowRussia
  3. 3.Division of Life Science (BK21 Program) and Environmental Biotechnology National Core Research CentreGraduate School of Gyeongsang National UniversityChinjuKorea

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